Studies on the callus induction and Agrobacterium-mediated transformation of Asia minor bluegrass (Polypogon fugax)

Asia Minor bluegrass (Polypogon fugax Nees ex Steud.) is one of the major grass weeds in winter crops in China, with populations having developed resistance to herbicides (such as clodinafop-propargyl). In this study, the methods for tissue culture and genetic transformation were established for this weedy species. Callus induction, adventitious bud differentiation and rooting were achieved by using mature seeds of clodinafop-propargyl-susceptible P. fugax, and by manipulating concentration combinations of 2,4-dichlorophenoxyacetic acid (2,4-D), 6-benzyl aminopurine (6-BA), and naphthaleneacetic acid (NAA). Genetic transformation was conducted by co-culturing generated calli with Agrobacterium tumefaciens EHA105 containing pOx (HA)-GFP plasmids, and regenerated transgenic plants were obtained by hygromycin screening. The green fluorescent protein (GFP) visual analysis showed GFP gene expression in transgenic calli, and RT-qPCR confirmed the stable expression of the exogenous hygromycin gene in P. fugax transgenic plants. Furthermore, Southern hybridization analysis confirmed the transgene integration. This transformation efficiency was 7%. These results indicated successful establishment of a reliable and efficient A. tumefaciens-mediated genetic transformation system, which will provide opportunities to better understand the evolution of herbicide resistance and the resistance genetics.